Calf rennet, obtained from the fourth stomach of unweaned calves, has traditionally been used as the coagulant for milk in the production of cheese. More recently, an enzyme produced during the fermentation of certain fungi has been found to be a suitable replacement for calf rennet, the supply of which is limited by the availability of calf stomachs.
While the milk clotting enzyme obtained from fungi (typically referred to as microbial rennet) is quite suitable for making cheese, it has a higher degree of thermal stability than calf rennet. This property is disadvantageous because the rennet ends up in the whey during the cheese making process, and residual rennet activity in the whey is undesirable. This presents no difficulty when calf rennet is used because it is thermally deactivated at normal pasteurization temperatures. This is not the case, however, with microbial rennet because of its greater thermal stability. Cornelius reports in U.S. Pat. No. 4,348,482 that the thermal stability of microbial rennet can be decreased without substantially reducing its milk clotting activity by contacting an aqueous solution thereof with a methionine-oxidizing means. This process has achieved significant acceptance in the marketplace, especially with microbial rennet from the fungus of the species Rhizomucor miehei (formerly Mucor miehei). The taxonomy of the genus Mucor was revised and both Mucor pusillus and Mucor meihei were reclassified into a new genus Rhizomucor because they were sufficiently different from other members of the genus Mucor to justify such reclassification.
Cornelius also reports in U.S. Pat. No. 4,362,818 that the milk coagulating activity of the microbial enzyme obtained from R. pusillus can be increased by acylating the enzyme with selected acid anhydrides, including maleic anhydride. This acylation typically results in an increase of about 50% in the enzyme's activity although in one run there is reported a product having 221% of its original activity after treatment with maleic anhydride.
Higashi et al report in U.S. Pat. No. 4,530,906 that the coagulating activity of microbial rennet from R. pusillus can be increased by treating it with succinic anhydride and that they have reported a low proteolytic activity/milk coagulating activity index for the treated enzyme.